Structural Dynamics and Catalytic Properties of a Multimodular Xanthanase

• Published in: ACS catalysis Vol. 8; no. 7; pp. 6021 - 6034
• Main Authors: Moroz, Olga V, Jensen, Pernille F, McDonald, Sean P, McGregor, Nicholas, Blagova, Elena, Comamala, Gerard, Segura, Dorotea R, Anderson, Lars, Vasu, Santhosh M, Rao, Vasudeva P, Giger, Lars, Sørensen, Trine Holst, Monrad, Rune Nygaard, Svendsen, Allan, Nielsen, Jens E, Henrissat, Bernard, Davies, Gideon J, Brumer, Harry, Rand, Kasper D, Wilson, Keith S
• Format: Journal Article
• Language: English
• Published: American Chemical Society 06.07.2018
• Subjects: Chemical and Process Engineering; Engineering Sciences; Food engineering; Life Sciences; enzyme; xanthan; enzyme dynamics; enzyme stability; hydrogen/deuterium exchange mass spectrometry; carbohydrate
• ISSN: 2155-5435; 2155-5435
• DOI: 10.1021/acscatal.8b00666

The precise catalytic strategies used for the breakdown of the complex bacterial polysaccharide xanthan, an increasingly frequent component of processed human foodstuffs, have remained a mystery. Here, we present characterization of an endo-xanthanase from Paenibacillus nanensis. We show that it is a CAZy family 9 glycoside hydrolase (GH9) responsible for the hydrolysis of the xanthan backbone capable of generating tetrameric xanthan oligosaccharides from polysaccharide lyase family 8 (PL8) xanthan lyase-treated xanthan. Three-dimensional structure determination reveals a complex multimodular enzyme in which a catalytic (α/α)6 barrel is flanked by an N-terminal “immunoglobulin-like” (Ig-like) domain (frequently found in GH9 enzymes) and by four additional C-terminal all β-sheet domains that have very few homologues in sequence databases and at least one of which functions as a new xanthan-binding domain, now termed CBM84. The solution-phase conformation and dynamics of the enzyme in the native calcium-bound state and in the absence of calcium were probed experimentally by hydrogen/deuterium exchange mass spectrometry. Measured conformational dynamics were used to guide the protein engineering of enzyme variants with increased stability in the absence of calcium; a property of interest for the potential use of the enzyme in cleaning detergents. The ability of hydrogen/deuterium exchange mass spectrometry to pinpoint dynamic regions of a protein under stress (e.g., removal of calcium ions) makes this technology a strong tool for improving protein catalyst properties by informed engineering.